Tyrosine kinases are a class of enzymes that catalyze phosphorylation of tyrosine residues of protein substrates via a transfer of the terminal phosphate of adenosine triphosphate. In many contexts, tyrosine kinases play critical roles in signal transduction for a number of cell functions, including cell proliferation, carcinogenesis, and cell differentiation.
EML4-ALK is a fusion-type protein tyrosine kinase that is present in ˜5% of non-small cell lung cancer (NSCLC) cases and which is generated as a result of a small inversion within the short arm of human chromosome 2 (Soda™, M. et al. (2007) Nature 448:561-566; Mano, H. (2008) Cancer Sci. 99:2349-2355). EML4-ALK undergoes constitutive dimerization as a result of interaction between the coiled-coil domain within the EML4 region of each monomer and thereby acquires pronounced oncogenic activity. Transgenic mice that express EML4-ALK, specifically in lung epithelial cells, develop hundreds of adenocarcinoma nodules in both lungs soon after birth, and oral administration of a specific inhibitor of ALK tyrosine kinase activity rapidly eradicates such nodules from the lungs (Soda, M. et al. (2008) Proc. Natl. Acad. Sci. USA 105:19893-19897). These observations reveal the essential role of EML4-ALK in the carcinogenesis of NSCLC harboring this fusion kinase, and they further support the feasibility of molecularly targeted therapy with ALK inhibitors for this cancer. For example, clinical trials of an inhibitor, PF-02341066, of the tyrosine kinase activity of both ALK and MET are under way for the treatment of EML4-ALK-positive NSCLC, and their interim results are promising (Kwak, E. L. et al. (2009) J. Clin. Oncol. 27(suppl):15s (abstract 3509)). A subset of EML4-ALK-positive tumors, however, do not respond to the inhibitor, with unknown molecular basis of treatment failure.
In addition to PF-02341066, other tyrosine kinase inhibitors (TKIs) have been shown to possess pronounced therapeutic activity in cancer patients. Imatinib mesylate, a TKI for ABL1 and KIT, for instance, markedly improves the outcome of individuals with chronic myeloid leukemia positive for the BCR-ABL1 fusion kinase or with a gastrointestinal stromal tumor positive for activated KIT (Druker, B. J. et al. (2001) N. Engl. J. Med. 344:1031-1037; Heinrich, M. C. et al. (2008) J. Clin. Oncol. 26:5360-5367). Furthermore, gefitinib and erlotinib, both of which are TKIs for the epidermal growth factor receptor (EGFR), are effective in the treatment of NSCLC associated with EGFR activation (Mok, T. S. et al. (2009) J. Clin. Oncol. 27:5080-2087; Mok, T. S. et al. (2009) N. Engl. J. Med. 361:947-957). Unfortunately, a subset of target tumors are either refractory to corresponding TKIs from the start of treatment or become resistant after an initial response. Secondary mutations in the target kinases that directly or allosterically affect the shape of the ATP-binding pocket, resulting in hindrance of TKI binding, have been detected in some cases of treatment failure (Deininger, M. et al. (2005) Blood 105:2640-2653; Kobayashi, S. et al. (2005) N. Engl. J. Med. 352:786-792; Pao, W. et al. (2005) PLoS Med. 2:e73; Shah, N. P. et al. (2002) Cancer Cell 2:117-125). Accordingly, there is an immediate need to identify mutations conferring resistance upon tyrosine kinases, such as EML4-ALK, in order to better develop compositions, kits, and methods for identifying, assessing, preventing, and treating disorders related to their aberrant expression and/or activity.